Method for identifying the length of the rice mesocotyl and SNP marker Kasp-7-13.7 used thereby and applications

Abstract

The application discloses a method for identifying the length of a rice mesocotyl and a SNP marker Kasp-7-13.7 used in the method and application. The application solves the technical problem of identifying or assisting in identifying the length of a rice mesocotyl. The application verifies that the length of the mesocotyl of the rice with the AA genotype at the 37th position of sequence 1 is longer than or longer than the rice with the GG genotype at the 37th position of sequence 1, and the average value of the mesocotyl of the rice variety with the GG homozygous type is reduced by 36.1% compared with the rice variety with the AA homozygous type, which indicates that the haplotype or genotype molecular marker can be used for rice molecular marker assisted selection breeding, and the selection efficiency of the length of the rice mesocotyl is significantly improved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a method for identifying the length of the rice mesocotyl and the SNP marker Kasp-7-13.7 used therein and its application. Background Technology

[0002] Compared to traditional seedling transplanting, direct-seeding rice offers significant water and labor savings, facilitates large-scale mechanization, and has the potential to achieve yields comparable to traditional transplanting. However, direct-seeding faces numerous challenges in actual field operations, including low emergence rates, inconsistent seedling growth, difficulty in weed control, and susceptibility to lodging. Among these challenges, the rice mesocotyl plays a crucial role, particularly during seedling emergence. Therefore, rice seedlings with longer mesocotyls have a competitive advantage over weeds. Identifying genes associated with mesocotyl elongation and selecting germplasm resources with longer mesocotyls is essential for promoting the widespread application of direct-seeding rice technology and reducing production costs. It is noteworthy that mesocotyl length is controlled by multiple genes and exhibits a complex quantitative trait. Significant differences in this characteristic exist among different rice varieties; therefore, identifying genes directly related to mesocotyl growth and developing closely linked molecular markers is one of the urgent problems to be solved in current hybridization breeding.

[0003] Single nucleotide polymorphism (SNP) marker technology plays an increasingly important role in constructing high-density genetic maps, locating quantitative trait genes, and detecting genotypes in germplasm resources, which has a positive impact on accelerating molecular breeding. By using molecular markers tightly linked to mesocotyl length trait loci (QTLs), mesocotyl length in specific varieties can be strategically enhanced within the framework of marker-assisted selection (MAS). This method has been widely applied in numerous fields such as linkage analysis, association analysis, marker-assisted selection, and crop design breeding. Furthermore, KASP (Kompetitive Allele-Specific PCR) technology, a rapid and accurate molecular detection method, can be used on a large scale to accurately identify SNPs and insertion / deletion variations (InDels) at specific sites in genomic DNA samples, thereby promoting the detection of specific markers in large numbers of samples. This technology improves the efficiency of the breeding process and is of great value for achieving more precise molecular breeding. Summary of the Invention

[0004] The technical problem solved by this invention is to provide a molecular marker for mesocotyl length.

[0005] To address the above problems, the present invention provides the following method.

[0006] A method for identifying or assisting in the identification of rice mesocotyl length includes detecting the genotype of an SNP in the rice to be tested, and identifying or assisting in the identification of the rice mesocotyl length based on the genotype of the rice to be tested: the rice mesocotyl length of the genotype AA is longer than or can be longer than the mesocotyl length of the genotype GG; the SNP site is a site on rice chromosome 7, and its nucleotide type is A or G, and it is the 37th nucleotide of sequence 1 in the sequence listing; AA is a homozygous type where the 37th nucleotide of SEQ ID No. 1 in the sequence listing is A; GG is a homozygous type where the 37th nucleotide of SEQ ID No. 1 in the sequence listing is G.

[0007] As one implementation scheme, the method for identifying or assisting in the identification of rice mesocotyl length may include the following steps:

[0008] (1) Using the genomic DNA of the rice to be tested as a template, KASP was performed using a primer set;

[0009] The primer set may include upstream primer F1, downstream primer F2, and downstream primer R;

[0010] The upstream primer F1 can be a single-stranded DNA molecule whose nucleotide sequence is position 22-52 of sequence 2 in the sequence listing;

[0011] The downstream primer F2 can be a single-stranded DNA molecule whose nucleotide sequence is position 22-52 of sequence 3 in the sequence listing.

[0012] The downstream primer R can be a single-stranded DNA molecule whose nucleotide sequence is sequence 4 in the sequence listing.

[0013] (2) After completing step (1), perform fluorescence detection to determine the genotype of the SNP in the rice to be tested;

[0014] (3) Identify or assist in identifying the length of the rice mesocotyl based on the genotype results: the genotype of the SNP is AA and the length of the rice mesocotyl of the test rice is longer than or can be longer than the length of the rice mesocotyl of the test rice with the genotype GG.

[0015] The application of the above methods in rice breeding.

[0016] The breeding can be for rice varieties with long or short mesocotyls.

[0017] The breeding can be for rice varieties with long mesocotyls.

[0018] To address the above problems, the present invention also provides the following applications.

[0019] The application is characterized in that the application is P1 or P2;

[0020] P1 refers to the application of substances used to detect SNP polymorphisms or genotypes in the identification or auxiliary identification of rice mesocotyl length, or their application in the preparation of products for the identification or auxiliary identification of rice mesocotyl length.

[0021] The P2 refers to the application of substances used to detect SNP polymorphisms or genotypes in rice breeding or the preparation of rice breeding products.

[0022] The SNP is the 37th nucleotide of SEQ ID No. 1 in the sequence listing, which is either A or G.

[0023] The substance may be a product. The detection substance may include reagents, kits, and instruments for detecting the above-mentioned single nucleotide polymorphisms. Specifically, it includes primers and other reagents and instruments required for in vitro nucleic acid amplification for detecting the above-mentioned single nucleotide polymorphisms.

[0024] The SEQ ID No. 1 is a part of the genomic sequence of the Kasp-7-13.7 gene. In actual detection, SNP polymorphism can be detected and analyzed by detecting the nucleotide polymorphism of the mRNA transcribed from the Kasp-7-13.7 gene, the cDNA reverse transcribed from the Kasp-7-13.7 mRNA, or the amino acid polymorphism of the Kasp-7-13.7 protein caused by SNP polymorphism.

[0025] In this application, the genotype of the SNP can be genotype AA or genotype GG, where genotype AA is homozygous for SNP A; and genotype GG is homozygous for SNP G.

[0026] The homozygous form of SNP A is the homozygous form of SEQ ID No. 1 in the sequence listing where the 37th position is nucleotide A.

[0027] The homozygous form of SNP G is the homozygous form of SEQ ID No. 1 in the sequence listing where the 37th position is nucleotide G.

[0028] To address the above problems, the present invention also provides the following products.

[0029] The product is characterized in that it contains any of the substances described above for detecting the polymorphism or genotype of rice genome SNPs, and is any one of the following G1)-G3):

[0030] G1) Products that detect single nucleotide polymorphisms or genotypes related to the length of the rice mesocotyl;

[0031] G2) Products for identifying or assisting in the identification of mesocoaxial length;

[0032] G3) is a product used in rice breeding.

[0033] In any of the above-described applications or products, the rice is a pure line of rice.

[0034] According to the above-mentioned applications, products, or applications or products, the purpose of the breeding includes cultivating or selecting rice varieties with short or long mesocotyl lengths.

[0035] The breeding objectives mentioned above include cultivating or selecting rice varieties with long mesocotyl lengths.

[0036] In the above-mentioned applications or products, the substance for detecting SNP polymorphisms or genotypes, or the substance for detecting haplotypes, is as follows: D1), D2), D3), or D4):

[0037] D1) Contains in vitro nucleic acid amplification primers that specifically amplify the SNP;

[0038] D2) An in vitro nucleic acid amplification reagent containing the in vitro nucleic acid amplification primers described in D1);

[0039] D3) A kit containing the in vitro nucleic acid amplification primers described in D1) or the in vitro nucleic acid amplification reagents described in D2);

[0040] D4) A detection instrument containing the in vitro nucleic acid amplification primers described in D1), the in vitro nucleic acid amplification reagents described in D2), or the kit described in D3).

[0041] The in vitro nucleic acid amplification technology may be polymerase chain reaction (PCR), chain substitution amplification (SDA), ligase chain reaction (LCR), sequence-dependent amplification (NASBA), rolling circle nucleic acid amplification (RCA), loop-mediated isothermal amplification (lamp), helicase-dependent isothermal amplification (HDA), or Qβ replication technology.

[0042] This application uses polymerase chain reaction (PCR) as an amplification method to detect polymorphism.

[0043] The specific amplification described in D1) can detect the nucleotide sequence of SNP polymorphic sites by the presence or absence of amplification products or by combining the presence or absence of amplification products with auxiliary reagents such as probes.

[0044] In the above applications, methods, and products, the in vitro nucleic acid amplification primers may or may not be labeled with a marker. The marker refers to any atom or molecule that can be used to provide a detectable effect and can be linked to a nucleic acid. Markers include, but are not limited to, dyes; radioactive labels, such as 32P; binding moieties, such as biotin; haptens, such as digoxigenin (DIG); luminescent, phosphorescent, or fluorescent moieties; and fluorescent dyes alone or in combination with moieties whose emission spectra can be inhibited or shifted by fluorescence resonance energy transfer (FRET). The marker can provide a signal detectable by fluorescence, radioactivity, colorimetry, gravimetric determination, X-ray diffraction or absorption, magnetism, enzyme activity, etc. The marker can be a charged moieties (positive or negative) or, optionally, charge-neutral. The marker can include nucleic acid or protein sequences or combinations thereof, provided that the sequence containing the marker is detectable. In some embodiments, nucleic acids are detected directly without labeling.

[0045] In the above applications, products, or methods, the in vitro nucleic acid amplification primers include upstream primer set F1, downstream primer F2, and downstream primer R:

[0046] The upstream primer F1 is a single-stranded DNA molecule whose nucleotide sequence is position 22-52 of sequence 2 in the sequence listing.

[0047] The downstream primer F2 is a single-stranded DNA molecule whose nucleotide sequence is position 22-52 of sequence 3 in the sequence listing.

[0048] The downstream primer R is a single-stranded DNA molecule whose nucleotide sequence is sequence 4 in the sequence listing.

[0049] The upstream primer F1 is an in vitro nucleic acid amplification primer for detecting SNP polymorphism site T, and can be a single-stranded DNA molecule whose nucleotide sequence is sequence 2 in the sequence listing (positions 1-21 of SEQ ID No. 2 in the sequence listing are FAM sequences).

[0050] The downstream primer F2 is an in vitro nucleic acid amplification primer for detecting SNP polymorphism site C, and can be a single-stranded DNA molecule whose nucleotide sequence is sequence 3 in the sequence listing (positions 1-21 of SEQ ID No. 3 in the sequence listing are FAM sequences).

[0051] In the above applications, products, and methods, the rice is a pure line.

[0052] In the above applications and methods, the product may be a reagent, kit, or system. The system may include a combination of reagents or kits, instruments, and analysis software, such as a product consisting of PCR primers, PARMS master mix reagents, an ELISA reader, and online SNP decoder software (http: / / www.snpway.com / snpdecoder01 / ), or a combination of PCR primers, PARMS master mix reagents, online SNP decoder software, and a real-time PCR instrument. The product may include the substances described above for detecting SNP polymorphisms or genotypes in the rice genome.

[0053] To address the aforementioned problems, the present invention also provides a method for rice breeding.

[0054] The method includes replacing the 37th nucleotide of sequence 1 in the genome of rice with genotype GG with A, to obtain rice with a longer mesocotyl than the rice with genotype GG; wherein GG is a homozygous type in which the 37th nucleotide of SEQ ID No. 1 in the sequence listing is G.

[0055] The method for replacing the 37th nucleotide of sequence 1 in the genome of rice with genotype GG with A includes the following steps: selecting rice with genotype AA and rice with genotype GG for hybridization, wherein AA is a homozygous type in which the 37th nucleotide of SEQ ID No. 1 in the sequence listing is A.

[0056] To address the aforementioned problems, the present invention also provides a method for breeding short mesocotyl rice.

[0057] The method includes replacing the 37th nucleotide of sequence 1 in the genome of rice with genotype AA with G, to obtain rice with a mesocotyl length shorter than that of rice with genotype AA and genotype GG; wherein AA is a homozygous type with nucleotide A at the 37th position of SEQ ID No. 1 in the sequence listing; and wherein GG is a homozygous type with nucleotide G at the 37th position of SEQ ID No. 1 in the sequence listing.

[0058] The plant genome with nucleotide A at position 37 of sequence 1 indicates a hypocotyl length longer than or candidate longer than that of the target plant genome with nucleotide G at position 37 of sequence 1.

[0059] The plant is a homozygous plant.

[0060] In the above applications, methods, and products, the substance may be a reagent and / or kit and / or instrument required to determine the polymorphism or genotype of the SNP by at least one of the following methods: in vitro nucleic acid amplification, DNA sequencing, restriction enzyme fragment length polymorphism, single-strand conformation polymorphism, denaturing high-performance liquid chromatography, and SNP chips. The SNP chips include chips based on nucleic acid hybridization reactions, chips based on single-base extension reactions, chips based on allele-specific primer extension reactions, chips based on one-step reactions, chips based on primer ligation reactions, chips based on restriction endonuclease reactions, chips based on protein-DNA binding reactions, and chips based on fluorescent molecule-DNA binding reactions.

[0061] In the applications and methods described above, rice varieties can be selected as parents for breeding.

[0062] The rice mesocotyl length mentioned above can specifically refer to the mesocotyl length 3 days after all seedlings have emerged from the soil.

[0063] The purpose of the breeding includes cultivating or selecting rice varieties with long or short mesocotyl lengths.

[0064] In the above applications and methods, the PCR primers may or may not be labeled with a marker. The marker refers to any atom or molecule that can be used to provide a detectable effect and can be linked to a nucleic acid. Markers include, but are not limited to, dyes; radioactive markers, such as 32P; binding moieties, such as biotin; haptens, such as digoxigenin (DIG); luminescent, phosphorescent, or fluorescent moieties; and fluorescent dyes alone or in combination with moieties whose emission spectra can be inhibited or shifted by fluorescence resonance energy transfer (FRET). The marker can provide a signal detectable by fluorescence, radioactivity, colorimetry, gravimetric determination, X-ray diffraction or absorption, magnetism, enzyme activity, etc. The marker can be a charged moiety (positive or negative charge) or, optionally, charge-neutral. The marker can include nucleic acid or protein sequences or combinations thereof, provided that the sequence containing the marker is detectable. In some embodiments, nucleic acids are detected directly without a marker (e.g., direct sequence reading).

[0065] In the above applications and methods, the product may be a reagent, kit, or system. The system may include a combination of reagents or kits, instruments, and analysis software, such as a product consisting of PCR primers, PARMS master mix reagents, an ELISA reader, and online SNP decoder software (http: / / www.snpway.com / snpdecoder01 / ), or a combination of PCR primers, PARMS master mix reagents, online SNP decoder software, and a real-time PCR instrument. The product may include the substances described above for detecting SNP polymorphisms or genotypes in the rice genome.

[0066] Beneficial effects

[0067] This invention utilizes 318 IR 145 / CHANGAI F 2:3 A polymorphic site associated with rice mesocotyl length was found in the population, namely, nucleotide 37 of SEQ ID No. 1 being either A or G. Two genotypes existed in the pure lines: genotype AA or genotype GG. Genotype AA is homozygous for SEQ ID No. 1 with nucleotide 37 being A, and genotype GG is homozygous for SEQ ID No. 1 with nucleotide 37 being G. The rice mesocotyl length of genotype AA is longer than or candidate to be longer than that of genotype GG.

[0068] Validation was conducted on 470 rice accessions. Two genotypes were found in the pure lines: AA and GG. Genotype AA is homozygous for position A at SEQ ID No. 1, and genotype GG is homozygous for position G at SEQ ID No. 1. Among the 470 rice varieties, the average mesocotyl length was 1.54 cm in the 250 AA genotypes and 1.00 cm in the 175 GG genotypes. The average mesocotyl length of the AA homozygous varieties (1.54 cm) was 36.1% lower than that of the GG homozygous varieties (1.00 cm). This indicates that haplotype or genotype molecular markers can be used for marker-assisted selection breeding of rice, significantly improving the selection efficiency for rice mesocotyl length. Attached Figure Description

[0069] Figure 1 This is a partial detection result diagram of the F2:3 population of 318 IR 145 and IR64 in Example 1; red represents the IR145 genotype (GG), blue represents the IR64 genotype (AA), pink represents detection failure, and green represents heterozygote (AG).

[0070] Figure 2This is a partial image of the detection results for 470 rice varieties in the Indic population in Example 2; red represents the IR145 genotype (GG), blue represents the IR64 genotype (AA), pink represents detection failure, and green represents heterozygote (AG). Detailed Implementation

[0071] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0072] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0073] The following examples used SPSS 11.5 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA was used. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, and P < 0.001 (***) indicates a highly significant difference.

[0074] 'IR 145' and IR64 are important indica rice varieties. IR 145 has a shorter mesocotyl, only 0.18 cm. Changai has a longer mesocotyl, 3.83 cm.

[0075] IR145 is described in the following literature: “QTL-seq and linkage analysis were combined to identify QTLs associated with rice mesocotyl elongation (named in this literature as the short mesocotyl variety ‘IR145’ or ‘IR145’)” and “Liu, J., Zhan, J., Chen, J., Lu, X., Zhi, S., & Ye, G. (2021). Validation of genes affecting rice grain zinccontent through candidate gene-based association analysis. Frontiers in Genetics, 12, 701658.

[0076] IR64 is described in the following literature: “QTL-seq and linkage analysis were combined to discover the hypocotyl elongation-related QTL in rice (named IR64 in this literature)” and “Mackill, DJ, & Khush, GS (2018). IR64: a high-quality and high-yielding mega variety. Rice, 11, 1-11.

[0077] Example 1: Correlation analysis and verification between Kasp-7-13.7 and the hypocotyl of natural rice varieties.

[0078] Kasp-7-13.7 is a SNP in the rice genome, specifically the 37th nucleotide of SEQ ID No. 1, which is either A or G. The genotype of Kasp-7-13.7 can be AA, AG, or GG. Genotype AA is homozygous for A in Kasp-7-13.7, genotype AG is heterozygous for both A and G in Kasp-7-13.7, and genotype GG is homozygous for G in Kasp-7-13.7.

[0079] 1. Genotyping of 470 rice varieties based on the KASP-7-13.7 locus.

[0080] The 470 rice varieties to be tested are recorded in the attachment of the following document: (Wang W, Mauleon R, Hu Z, Chebotarov D, Tai S, Wu Z, Li M, Zheng T, Fuentes RR, Zhang F, Mansueto L, Copetti D, Sanciangco M, Palis KC, Xu J, Sun C, Fu B, Zhang H, Gao Y, Zhao X, Shen F, Cui X,YuH,Li Z,Chen M,Detras J,Zhou Y,Zhang X,Zhao Y,Kudrna S,Wang C,Li R,Jia B,Lu J,He J,Li J,Gao Q,Niu Y,Yue Z,Naredo M EB,Talag J,Wang X,Li J,Fang X,Yin Y,Glaszmann JC,Zhang J,Li J,Hamilton RS,Wing R A,Ruan J,Zhang G,Wei C, Alexandrov N,McNally K L,Li Z,Leung H. Genomic variation in 3,010 diverse accessions of Asian cultivated rice. Nature, 2018, 557:43-49 (specific variety information is shown in Table 1).

[0081] Molecular identification:

[0082] (1) Genomic DNA was extracted from young leaves of 470 rice germplasm resources using the CTAB method. The quality and concentration of the genomic DNA had to meet the requirements of PCR, namely: agarose gel electrophoresis showed a single DNA band, an A260 / A280 ratio between 1.8 and 2.0, an A260 / A230 ratio between 1.8 and 2.0, and no obvious light absorption at 270 nm; the concentration of the genomic DNA of the rice to be tested was 50-200 ng / μL.

[0083] (2) Competitive allele-specific PCR. Using the genomic DNA of the rice to be tested as a template, PCR amplification was performed using the primer set synthesized in Example 2 (composed of upstream primer F1 (FAM), upstream primer F2 (HEX), and downstream primer R) to obtain PCR amplification products. The reaction program was as follows: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, 61℃-55℃ (touch down program, decreasing by 0.6℃ per cycle) for 1 min, amplification for 10 cycles; 94℃ denaturation for 20 s, 55℃ for 1 min, continued amplification for 26 cycles.

[0084] (3) After completing step (2), when the temperature of the PCR amplification product drops below 40℃, the fluorescence value is read by scanning the FAM and HEX beams of the microplate reader (the FAM fluorescent tag sequence is read at an excitation wavelength of 485nm and an emission wavelength of 520nm, and the HEX fluorescent tag sequence is read at an excitation wavelength of 528nm and an emission wavelength of 560nm). The genotype of the rice to be tested based on the Kasp-7-13.7 site is determined according to the fluorescence signal color.

[0085] The specific judgment principles are as follows: If the tested rice shows a red fluorescent signal based on the Kasp-7-13.7 locus, then the genotype of the tested rice based on the Kasp-7-13.7 locus is GG homozygous, consistent with IR145; if the tested rice shows a blue fluorescent signal based on the Kasp-7-13.7 locus, then the genotype of the tested rice based on the Kasp-7-13.7 locus is AA homozygous, consistent with IR64; if the tested rice shows a green fluorescent signal based on the Kasp-7-13.7 locus, then the genotype of the tested rice based on the Kasp-7-13.7 locus is AG heterozygous. Based on the above method, the genotypes of 470 rice varieties based on the KASP-7-13.7 locus were obtained. The detection results are shown below. Figure 2 And Table 4.

[0086] 2. Detection of the mesocotyl

[0087] 1. 470 rice accessions from the Indica subgroup in Southeast Asia and South Asia were selected. From each accession, 15 plump, uncracked rice grains were selected and sown in 10×5-well trays containing a measured amount of homogeneous potting soil, one variety per cell. Ensure a sowing depth of 6 cm. Cover with potting soil until level with the surface of the cell. Weigh 500g of potting soil into the tray, spray the cell and tray separately with tap water, and place the entire setup in a 30℃ constant temperature dark incubator. Water regularly daily until seedling emergence, and record germination status. After approximately 10 days of constant temperature incubation, remove the cell trays from the artificial climate chamber, quickly rinse the soil around the seedling roots with running water, and remove any individual plants with significantly different growth patterns. Photograph the uniformly growing lines and measure the mesocotyl length using ImageJ software (https: / / imagej.en.softonic.com / ).

[0088] 3. Correlation Analysis

[0089] The average mesocotyl length of rice from the two genotypes was statistically analyzed, and a t-test was performed using the PROCTTEST model in the internationally used SAS 9.2 statistical software. The results are shown in Table 4. The results showed that the average mesocotyl length of the GG homozygous rice variety (mesocotyl length 1.00 cm) was 36.1% lower than that of the AA homozygous rice variety (mesocotyl length 1.54 cm), which was statistically significant at the P=0.05 level (Table 5).

[0090] Table 4. Genotyping results of 470 rice varieties

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105]

[0106]

[0107] Red indicates the IR145 genotype (GG), blue indicates the IR64 genotype (AA), "-" indicates a failed test, and green indicates heterozygote (AG).

[0108] Table 5. Correlation analysis of mesocotyl length in 470 Indica samples (Kasp-7-13.7)

[0109]

[0110] The above results indicate that rice mesocotyls can be identified by detecting the genotype of the rice sample based on the Kasp-7-13.7 locus, which has important application value in the process of molecular marker-assisted breeding of rice.

[0111] Example 2: Development and Polymorphism Detection of Kasp-7-13.7, a Molecular Marker for Mesocotyl Length in Rice

[0112] Development of a molecular marker, Kasp-7-13.7

[0113] Example 1 is the verification of the molecular marker Kasp-7-13.7 for the embryonic axis length. The present invention found the molecular marker Kasp-7-13.7 through the following experiments, as follows:

[0114] Through extensive experimentation, the inventors of this invention designed and synthesized a set of primers suitable for identifying rice mesoderms using allele-competitive specific PCR on rice chromosome 7 (13.7 Mb) via fine mapping. A molecular marker related to mesoderm length, Kasp-7-13.7, was discovered. Kasp-7-13.7 is a SNP in the rice genome, located at nucleotide 37 from the 5' end of SEQ ID NO: 1 in the rice genome. The SNP genotypes are AA, GG, and AG.

[0115] The AA type (AA homozygous) is a homozygous type with A at position 37 of SEQ ID NO: 1 in the genome; the GG type (GG homozygous) is a homozygous type with G at position 37 of SEQ ID NO: 1 in the genome; and the AG type (AG heterozygous) is a heterozygous type with both A and G at position 37 of SEQ ID NO: 1 in the genome.

[0116] SEQ ID NO: 1: CCATAGAAACCCAGATAATTTAGTATTGCTTACTCTrCTTATAATCTGGTTAAAATATCT TATACTTAGATTG, where r is A or G.

[0117] A primer set was designed, consisting of upstream primer F1 (FAM), upstream primer F2 (HEX), and downstream primer R, to amplify the target sequence including the Kasp-7-13.7 site. The nucleotide sequences of each primer are shown in Table 1.

[0118] Table 1. Nucleotide sequences of each primer

[0119]

[0120]

[0121] The underlined sequence in upstream primer F1 is the FAM sequence tag sequence, and the underlined sequence in upstream primer F2 is the HEX sequence tag sequence.

[0122] Since genomic DNA is composed of two single-stranded DNA molecules that are antisense complements each other, the DNA molecule that encodes proteins is generally called the sense DNA molecule; the DNA molecule that is antisense complement to the sense DNA molecule is called the antisense DNA molecule. The genotypes at the Kasp-7-13.7 loci are all sense DNA genotypes.

[0123] Polymorphism detection

[0124] 1. Phenotypic identification of field mesocotyls of IR 145 / IR64

[0125] (1) Using rice IR145 as the female parent and rice IR64 as the male parent, after harvesting F1 generation seeds, the F1 generation was introduced for breeding in Lingshui County, Hainan Province in December to construct the F1 generation. 2:3 Population. From the IR 145 / IR64 population, 400 plump, uncracked rice grains were selected (due to some grains not germinating or poor seedling condition, phenotypic data from 318 plant accessions were ultimately used). These were sown in 10×5-well trays containing a measured amount of homogeneous potting soil, ensuring a sowing depth of 6 cm. Ensure no grains were pressed together, and cover with potting soil until level with the surface of the holes. Weigh 500g of potting soil into the tray and compact it. Spray the seedling tray and the potting soil with an appropriate amount of tap water, and place the entire setup in a 30℃ constant temperature dark incubator. Water regularly until seedlings emerge, and record germination status. After approximately 10 days of constant temperature cultivation, the seedling trays were removed from the artificial climate chamber. The soil around the seedling roots was quickly rinsed with running water, and individual plants with significant differences in growth were removed. The uniformly growing lines were photographed, and the mesocotyl length was measured using ImageJ software (https: / / imagej.en.softonic.com / ). The results are shown in Table 2. The t-test was performed using the PROC TTEST model of the internationally used SAS 9.2 statistical software. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, and P < 0.001 (***) indicates a highly significant difference. The results are shown in Table 3.

[0126] 2318 rice F 2:3 Molecular identification of populations (molecular identification methods)

[0127] (1) Genomic DNA was extracted from young leaves of 318 rice germplasm resources using the CTAB method. The quality and concentration of genomic DNA had to meet the requirements of PCR, namely: agarose gel electrophoresis showed a single DNA band, an A260 / A280 ratio between 1.8 and 2.0, an A260 / A230 ratio between 1.8 and 2.0, and no obvious light absorption at 270 nm; the concentration of genomic DNA of the rice samples was 50-200 ng / μL.

[0128] (2) Competitive allele-specific PCR. Using the genomic DNA of the rice to be tested as a template, PCR amplification was performed using the primer set synthesized above (consisting of upstream primer F1 (FAM), upstream primer F2 (HEX), and downstream primer R3). The reaction program was as follows: 94℃ pre-denaturation for 15 min; 94℃ denaturation for 20 s, 61℃-55℃ (touch-down program, decreasing by 0.6℃ per cycle) for 1 min, for 10 cycles; 94℃ denaturation for 20 s, 55℃ for 1 min, for 26 more cycles.

[0129] (3) After completing step (2), when the temperature of the PCR amplification product drops below 40℃, the fluorescence value is read by scanning the FAM and HEX beams of the microplate reader (the FAM fluorescent tag sequence is read at an excitation wavelength of 485nm and an emission wavelength of 520nm, and the HEX fluorescent tag sequence is read at an excitation wavelength of 528nm and an emission wavelength of 560nm). The genotype of the rice to be tested based on the Kasp-7-13.7 site is determined according to the fluorescence signal color.

[0130] The specific judgment principles are as follows: If the tested rice shows a red fluorescent signal at the Kasp-7-13.7 locus, then the genotype of the tested rice at the Kasp-7-13.7 locus is GG homozygous, consistent with IR145; if the tested rice shows a blue fluorescent signal at the Kasp-7-13.7 locus, then the genotype of the tested rice at the Kasp-7-13.7 locus is AA homozygous, consistent with IR64; if the tested rice shows a green fluorescent signal at the Kasp-7-13.7 locus, then the genotype of the tested rice at the Kasp-7-13.7 locus is AG heterozygous. The test results are shown below. Figure 1 And Table 2.

[0131] III. Significance Analysis

[0132] The average mesocotyl length of the two rice genotypes was statistically analyzed (Table 2), and a t-test was performed using the PROC TTEST model in the internationally used SAS 9.2 statistical software. The statistical results are shown in Table 3. The results showed that the average mesocotyl length of the GG homozygous rice variety (mesocotyl 1.12 cm) was 25.8% lower than that of the AA homozygous rice variety (mesocotyl 1.51 cm), which was statistically significant at the 0.05 level (Table 3).

[0133] Therefore, the genotype at the Kasp-7-13.7 locus can be used to identify the mesocotyl of rice. The criteria are as follows: if the rice genotype at the Kasp-7-13.7 locus is homozygous AA, the rice mesocotyl is relatively long; if the rice genotype at the Kasp-7-13.7 locus is homozygous GG, the rice mesocotyl is relatively short.

[0134] Table 2. KASP-7-13.7 detection results and mesocotyls in the IR 145 / IR64 F2:3 population.

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146] IR64 genotype (AA), IR145 genotype (GG), "-" indicates deletion, green indicates heterozygote (AG).

[0147] Table 3. Mesocotyl association analysis of Kasp-1-38383221 in the IR 145 / IR64 population.

[0148]

[0149] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for identifying or assisting in the identification of rice mesocotyl length, comprising detecting the genotype of an SNP in the rice to be tested, and identifying or assisting in the identification of the rice mesocotyl length based on the genotype of the rice to be tested: the rice mesocotyl length of the genotype AA is longer than the mesocotyl length of the genotype GG; the SNP site is a site on rice chromosome 7, the nucleotide type is A or G, and it is the 37th nucleotide of SEQ ID No. 1 in the sequence listing; AA is a homozygous type where the 37th nucleotide of SEQ ID No. 1 in the sequence listing is A; and GG is a homozygous type where the 37th nucleotide of SEQ ID No. 1 in the sequence listing is G.

2. The application of the method of claim 1 in rice breeding, wherein the purpose of the breeding includes cultivating or selecting rice varieties with short or long mesocotyl lengths.

3. Application, characterized in that, The application is P1 or P2; The P1 refers to the application of substances that detect SNP polymorphisms or genotypes in the identification or auxiliary identification of rice mesocotyl length, or in the preparation of products for the identification or auxiliary identification of rice mesocotyl length. The P2 refers to the application of substances for detecting SNP polymorphisms or genotypes in rice breeding or the preparation of rice breeding products. The purpose of the breeding includes cultivating or selecting rice varieties with short or long mesocotyl lengths. The substance used to detect SNP polymorphisms or genotypes is D1), D2), D3), or D4): D1) Contains in vitro nucleic acid amplification primers that specifically amplify the SNP; D2) An in vitro nucleic acid amplification reagent containing the in vitro nucleic acid amplification primers described in D1); D3) A kit containing the in vitro nucleic acid amplification primers described in D1) or the in vitro nucleic acid amplification reagents described in D2); D4) A detection instrument containing the in vitro nucleic acid amplification primers described in D1), the in vitro nucleic acid amplification reagents described in D2), or the kit described in D3); The SNP is the 37th nucleotide of SEQ ID No. 1 in the sequence listing, which is either A or G.

4. The application according to claim 2 or 3, characterized in that, The rice variety in question is a pure line of rice.

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